This is the report of the analysis made for the paper (TITLE) AND AUTHORS. INSERT ABSTRACT
Importing data and filtering out those genes with cpm lesser than 1. We use the filtered.data method in NOISeq package.
countMatrix <- ReadDataFrameFromTsv(file.name.path="../../data/refSEQ_countMatrix.txt")
# head(countMatrix)
designMatrix <- ReadDataFrameFromTsv(file.name.path="../../design/all_samples_short_names_noRS2HC7.tsv")
# head(designMatrix)
filteredCountsProp <- filterLowCounts(counts.dataframe=countMatrix,
is.normalized=FALSE,
design.dataframe=designMatrix,
cond.col.name="gcondition",
method.type="Proportion")
## Filtering out low count features...
## 14454 features are to be kept for differential expression analysis with filtering method 3
Loading Negative Control Genes to normalize data
library(readxl)
sd.ctrls <- read_excel(path="../../data/controls/Additional File 4 full list of BMC genomics SD&RS2.xlsx", sheet=1)
sd.ctrls <- sd.ctrls[order(sd.ctrls$adj.P.Val),]
sd.neg.ctrls <- sd.ctrls[sd.ctrls$adj.P.Val > 0.9, ]
sd.neg.ctrls <- sd.neg.ctrls$`MGI Symbol`
sd.neg.ctrls <- sd.neg.ctrls[-which(is.na(sd.neg.ctrls))]
int.neg.ctrls <- sd.neg.ctrls
int.neg.ctrls <- unique(int.neg.ctrls)
neg.map <- convertGenesViaMouseDb(gene.list=int.neg.ctrls, fromType="SYMBOL",
"ENTREZID")
# sum(is.na(neg.map$ENTREZID))
neg.ctrls.entrez <- as.character(neg.map$ENTREZID)
ind.ctrls <- which(rownames(filteredCountsProp) %in% neg.ctrls.entrez)
counts.neg.ctrls <- filteredCountsProp[ind.ctrls,]
Loading Positive Control Genes to detect them during the differential expression step.
## sleep deprivation
sd.lit.pos.ctrls <- read_excel("../../data/controls/SD_RS_PosControls_final.xlsx",
sheet=1)
colnames(sd.lit.pos.ctrls) <- sd.lit.pos.ctrls[1,]
sd.lit.pos.ctrls <- sd.lit.pos.ctrls[-1,]
sd.est.pos.ctrls <- read_excel("../../data/controls/SD_RS_PosControls_final.xlsx",
sheet=3)
sd.pos.ctrls <- cbind(sd.est.pos.ctrls$`MGI Symbol`, "est")
sd.pos.ctrls <- rbind(sd.pos.ctrls, cbind(sd.lit.pos.ctrls$Gene, "lit"))
sd.pos.ctrls <- sd.pos.ctrls[-which(duplicated(sd.pos.ctrls[,1])),]
sd.pos.ctrls <- sd.pos.ctrls[-which(is.na(sd.pos.ctrls[,1])),]
Normalizing data with TMM, as implemented in edgeR package, and applying a RUVs method of RUVSeq package on normalized data, in order to adjust the counts for the unwanted variation. We plot a PCA and an RLE plot on these data.
normPropCountsUqua <- NormalizeData(data.to.normalize=filteredCountsProp,
norm.type="tmm",
design.matrix=designMatrix)
neg.ctrl.list <- rownames(counts.neg.ctrls)
groups <- makeGroups(designMatrix$gcondition)
ruvedSExprData <- RUVs(as.matrix(round(normPropCountsUqua)), cIdx=neg.ctrl.list,
scIdx=groups, k=5)
normExprData <- ruvedSExprData$normalizedCounts
ggp <- PlotPCAPlotlyFunction(counts.data.frame=log1p(normExprData),
design.matrix=designMatrix, shapeColname="condition",
colorColname="genotype", xPCA="PC1", yPCA="PC2",
plotly.flag=FALSE, show.plot.flag=FALSE, save.plot=FALSE,
prefix.plot=NULL)
## [1] FALSE
ggplotly(ggp)
dir.create("plots")
save_plot(filename="plots/PCA.pdf", plot=ggp)
pal <- RColorBrewer::brewer.pal(9, "Set1")
plotRLE(normExprData, outline=FALSE, col=pal[designMatrix$gcondition])
Making differential expression analysis with edgeR package on four different contrasts.
Here is a brief legend:
padj.thr <- 0.05
venn.padgj.thr <- 0.1
desMat <- cbind(designMatrix, ruvedSExprData$W)
colnames(desMat) <- c(colnames(designMatrix), colnames(ruvedSExprData$W))
cc <- c("S3HC5 - WTHC5", "S3SD5 - WTSD5")
rescList1 <- applyEdgeR(counts=filteredCountsProp, design.matrix=desMat,
factors.column="gcondition",
weight.columns=c("W_1", "W_2", "W_3", "W_4", "W_5"),
contrasts=cc, useIntercept=FALSE, p.threshold=1,
is.normalized=FALSE, verbose=TRUE)
names <- names(rescList1)
rescList1 <- lapply(seq_along(rescList1), function(i)
{
attachMeans(normalized.counts=normExprData, design.matrix=desMat,
factor.column="gcondition", contrast.name=names(rescList1)[i],
de.results=rescList1[[i]])
})
names(rescList1) <- names
A volcano plot of differential expressed genes.
res.o.map1 <- convertGenesViaMouseDb(gene.list=rownames(rescList1[[1]]),
fromType="ENTREZID")
res.o <- attachGeneColumnToDf(mainDf=rescList1[[1]],
genesMap=res.o.map1,
rowNamesIdentifier="ENTREZID",
mapFromIdentifier="ENTREZID",
mapToIdentifier="SYMBOL")
WriteDataFrameAsTsv(data.frame.to.save=res.o,
file.name.path=paste0(names(rescList1)[1], "_edgeR"))
vp <- luciaVolcanoPlot(res.o, prefix=names(rescList1)[1],
positive.controls.df=NULL,
threshold=padj.thr)
ggplotly(vp)
de <- sum(res.o$FDR < padj.thr)
nde <- sum(res.o$FDR >= padj.thr)
detable <- cbind(de,nde)
rownames(detable) <- names(rescList1)[1]
ddetable <- detable
tot.ctrls <- dim(sd.pos.ctrls)[1]
idx.pc <- which(tolower(res.o$gene) %in% tolower(sd.pos.ctrls[,1]))
tot.pc.de <- sum(res.o$FDR[idx.pc] < padj.thr)
tot.pc.nde <- length(idx.pc) - tot.pc.de
wt <- res.o[which(res.o$FDR < padj.thr),]
wt.sign.genes.entrez <- rownames(res.o)[which(res.o$FDR < venn.padgj.thr)]
kowthc5 <- res.o[which(res.o$FDR < padj.thr),]
kowthc5.sign.genes.entrez <- rownames(res.o)[which(res.o$FDR < venn.padgj.thr)]
A volcano plot of differential expressed genes.
rs2.o.map <- convertGenesViaMouseDb(gene.list=rownames(rescList1[[2]]),
fromType="ENTREZID")
res.rs2.o <- attachGeneColumnToDf(mainDf=rescList1[[2]],
genesMap=rs2.o.map,
rowNamesIdentifier="ENTREZID",
mapFromIdentifier="ENTREZID",
mapToIdentifier="SYMBOL")
WriteDataFrameAsTsv(data.frame.to.save=res.rs2.o,
file.name.path=paste0(names(rescList1)[2], "_edgeR"))
vp <- luciaVolcanoPlot(res.rs2.o, positive.controls.df=sd.pos.ctrls,
prefix=names(rescList1)[2],
threshold=padj.thr)
ggplotly(vp)
de <- sum(res.rs2.o$FDR < padj.thr)
nde <- sum(res.rs2.o$FDR >= padj.thr)
detable <- cbind(de,nde)
rownames(detable) <- names(rescList1)[2]
ddetable <- rbind(ddetable, detable)
pos.df <- cbind(tot.ctrls, tot.pc.de, tot.pc.nde)
colnames(pos.df) <- c("total_p.ctrl", "p.ctrl_de_mapped",
"p.ctrl_notde_mapped")
rownames(pos.df) <- names(rescList1)[2]
kowtsd5 <- res.rs2.o[which(res.rs2.o$FDR < padj.thr),]
kowtsd5.sign.genes.entrez <- rownames(res.rs2.o)[which(res.rs2.o$FDR < venn.padgj.thr)]
We present a summarization of the results. The first table is a summarization on how many genes are Differentially Expressed. The second table explains on the first column how many positive controls we have, on the second column how many positive controls have been identified over the differentially expressed genes, and, finally, on the third column how many positive controls have beed identified on the NOT differentially expressed genes.
ddetable
## de nde
## S3HC5 - WTHC5 39 14415
## S3SD5 - WTSD5 82 14372
pos.df
## total_p.ctrl p.ctrl_de_mapped p.ctrl_notde_mapped
## S3SD5 - WTSD5 579 3 553
We take the results of the two contrasts. Shank3 Sleed Deprivation VS Wild Type Sleep Deprivation and Shank3 Home Cage control VS Wild Type Home Cage Controls. And plot the results in a Venn Diagram
source("../../R/venn2.R")
gene.map <- convertGenesViaMouseDb(gene.list=rownames(normExprData),
fromType="ENTREZID", toType="SYMBOL")
venn <- Venn2de(x=kowthc5.sign.genes.entrez, y=kowtsd5.sign.genes.entrez,
label1="S3HC5_WTHC5", label2="S3SD5_WTSD5",
title="S3HC5_WTHC5 venn S3SD5_WTSD5", plot.dir="./",
conversion.map=gene.map)
Setting up the data structures for the heatmap.
source("../../R/heatmapFunctions.R")
de.genes.entr <- union(rownames(venn$int), rownames(venn$XnoY))
de.genes.entr <- union(de.genes.entr, rownames(venn$YnoX))
gene.map <- convertGenesViaMouseDb(gene.list=de.genes.entr,
fromType="ENTREZID")
de.genes.symb <- attachGeneColumnToDf(as.data.frame(de.genes.entr,
row.names=de.genes.entr),
genesMap=gene.map,
rowNamesIdentifier="ENTREZID",
mapFromIdentifier="ENTREZID",
mapToIdentifier="SYMBOL")
# de.genes.symb[which(is.na(de.genes.symb$gene)),]
de.genes.symb$gene[which(de.genes.symb$de.genes.entr=="100039826")] <- "Gm2444" ## not annotated in ncbi
de.genes.symb$gene[which(de.genes.symb$de.genes.entr=="210541")] <- "Entrez:210541" ## not annotated in ncbi
de.genes.counts <- normExprData[match(de.genes.symb$de.genes.entr, rownames(normExprData)),]
rownames(de.genes.counts) <- de.genes.symb$gene
de.gene.means <- computeGeneMeansOverGroups(counts=de.genes.counts,
design=designMatrix, groupColumn="gcondition")
library(gplots)
library(clusterExperiment)
color.palette = clusterExperiment::seqPal3#c("black", "yellow")
pal <- colorRampPalette(color.palette)(n = 1000)
library(pheatmap)
filter2 <- rowMeans(de.gene.means)>0
filter <- apply(de.gene.means, 1, function(x) log(x[4]/x[3]) * log(x[2]/x[1]) < 0)
filter[is.na(filter)] <- FALSE
idx <- which(!(rownames(de.genes.counts) %in% c("Nr1d1", "Fabp7", "Per3",
"Jun", "Elk1", "Fosl2", "Mapk1", "Mapk3", "Mapk11")))
de.genes.counts1 <- de.genes.counts
rownames(de.genes.counts1)[idx] <- ""
ann.col <- desMat[, c(1:2)]
de.heatmap <- de.genes.counts[filter2,]
set.seed(0)
ph1 <- pheatmap(log(de.heatmap+1), cluster_cols=TRUE, scale="row",
color=pal, border_color=NA, fontsize_row=10, kmeans_k=4, annotation_col=ann.col,
silent=TRUE)
clusterized.genes <- as.data.frame(ph1$kmeans$cluster)
gene.map <- convertGenesViaMouseDb(gene.list=rownames(clusterized.genes), fromType="SYMBOL")
converted.clusterized.gens <- attachGeneColumnToDf(mainDf=clusterized.genes, genesMap=gene.map,
rowNamesIdentifier="SYMBOL", mapFromIdentifier="SYMBOL", mapToIdentifier="ENTREZID")
converted.clusterized.gens$gene[which(rownames(converted.clusterized.gens)=="Gm2444")] <- "100039826" ## not annotated in ncbi
converted.clusterized.gens$gene[which(rownames(converted.clusterized.gens)=="Entrez:210541")] <- "210541" ## not annotated in ncbi
converted.clusterized.gens <- converted.clusterized.gens[order(converted.clusterized.gens$`ph1$kmeans$cluster`),]
ord.de.genes.counts <- de.heatmap[match(rownames(converted.clusterized.gens), rownames(de.heatmap)),]
idx <- which(!(rownames(ord.de.genes.counts) %in% c("Nr1d1", "Fabp7", "Per3",
"Jun", "Elk1", "Fosl2", "Mapk1", "Mapk3", "Mapk11")))
rownames(ord.de.genes.counts)[idx] <- ""
gaps.row <- c()
for(i in c(1:4))
{
li <- length(which(converted.clusterized.gens$`ph1$kmeans$cluster`==i))
l <- ifelse(i!=1, gaps.row[i-1]+li, li)
gaps.row <- c(gaps.row, l)
}
ph1 <- pheatmap(log(ord.de.genes.counts+1), cluster_cols=TRUE, scale="row",
color=pal, border_color=NA, fontsize_row=9, fontsize_col=9, cluster_rows=FALSE,
annotation_col=ann.col, gaps_row=gaps.row)
save_pheatmap_pdf(filename="plots/heatmap_gg_k4.pdf", plot=ph1, width=20, height=20)
## png
## 2
g <- geneGroupProfileRows(normalized.counts=normExprData, design.matrix=designMatrix,
gene.names=c("Nr1d1", "Fabp7", "Per3"),
res.o=de.genes.symb, show.plot=TRUE, plotly.flag=FALSE, log.flag=TRUE)
ggplotly(g)
save_plot(filename=paste0("plots/", "Nr1d1_Fabp7_Per3", "_log_gene_profile_genotype.pdf"), plot=g,
base_height=15, base_width=15)
g <- geneGroupProfileRows(normalized.counts=normExprData, design.matrix=designMatrix,
gene.names=c("Jun", "Elk1", "Fosl2", "Mapk1", "Mapk3", "Mapk11"),
res.o=de.genes.symb, show.plot=TRUE, plotly.flag=FALSE, log.flag=TRUE)
ggplotly(g)
save_plot(filename=paste0("plots/", "Jun_Elk1_Fosl2_Mapk", "_log_gene_profile_genotype.pdf"), plot=g,
base_height=15, base_width=15)